The invention relates to sample carriers and methods for cryopreservation of biological samples.
In modern biomedicine and biotechnology there is a continually growing need for cryopreserved biological materials, especially cells, cell groups, natural or artificial tissue, whole organs and cells of embryonic organisms. Cryopreservation is understood as the cooling of biological materials to low, so-called cryogenic temperatures, especially in the range from −80° C. to −196° C. Under these conditions, the metabolism of living cells comes almost to a standstill so that the cells can be stored over many years.
In order to preserve the vitality of cryopreserved cells, special cryomedia are used, comprising protective substances (cryoprotectants) such as dimethyl sulphoxide (DMSO), ethylene glycol, glycerol and sugars such as trehalose or glucose, for example. Cryoprotectants protect the cells by stabilising the cell membranes and macromolecules and by inhibiting intracellular ice formation. The intracellular ice crystals are primarily responsible for irreversible mechanical damage to the cell membranes (see S. S. N. Murthy in “Cryobiology”, vol. 36, 1998, p. 84-96). The optimal composition of the cryomedium is experimentally matched to the respective cell or tissue type.
In addition to the composition of the cryomedium, the freezing and thawing profile (rate, temperature gradients, duration etc.) have an essential role for the preservation of high cell vitalities. Numerous protocols for various types of samples and preservation conditions have been published which describe both fast and slow cooling and thawing rates or a combination of the two (see, for example, J. M. Baust et al. in “Cell Transplantation”, vol. 10, 2001, p. 561-571, and M. Miyamoto et al., in “Cell Transplantation” vol. 10, 2001, p. 363-371). It has been shown that a precisely controlled temperature regulation is required in each case since the cell vitality depends very strongly on the cooling/thawing rate.
If cell suspensions or tissue are frozen in conventional plastic cryovessels, it is not guaranteed that the same cooling rate is precisely maintained over all cells in the sample. Contributory factors to this are on the one hand the unfavorable surface/volume ratio (cryovessel/cell sample) and on the other hand, the low thermal conductivities of the conventionally used plastic of the cryovessels and the aqueous cryomedium.
This not only results in a steep temperature gradient inside the frozen sample but also in a strong spatial inhomogeneity of the cooling rate. The individual cells experience different temperature conditions, cooling and thawing rates according to their position in the sample.
This strong inhomogeneity in the cooling rates for the individual cells thus results in severe losses in the vitality since only the provision of the optimal cooling rate at all cells results in a high vitality of the sample.
In order to avoid and eliminate these problems, the following solution attempts are known. When freezing pig sperm cells in flat plastic containers (so-called flat-packs), B. M. Eriksson et al. (see “Anim. Reprod. Sci.” vol. 63, 2000, p. 205-220) were able to demonstrate a significant increase in the mobility of the sperm cells compared to freezing in cylindrical plastic tubes (so-called maxi-straws). The authors gave the reasons for this result as a more uniform freezing and thawing process in the flat cryovessels. Also problematical is a severely reduced thawing rate in the core of the samples in the “maxi-straws” as a result of the insulating properties of the already thawed water in the periphery of the samples.
S.-P. Park et al. (see “Human Reproduction” vol. 15, 2000, p. 1787-1790) describe the ultra-fast freezing of human embryos on copper lattices which were originally designed for electron microscopy. In this case, very rapid heat removal takes place when the samples are inserted in liquid nitrogen, which is again beneficial to the vitality of the embryos.
A commercially available system for the storage of micro-organisms is known from practice (product “micrybank”, manufacturer: SensLab-GmbH, Leipzig, Germany). Micro-organisms are bound to the porous surface of ceramic spheres which are preserved in a special cryomedium. This system however merely serves for improved handling of the samples. The freezing process is not optimised. The ceramic spheres have a lower thermal conductivity so that rapid freezing or thawing is impeded. In addition, because of their size, cells or tissue are not suited for adsorption on the porous surfaces.
It is also known to freeze biological samples in the form of very small sample volumes (ml or μl range) on suitably structured, two-dimensional cryosubstrates. In this case, said problems of temperature and cooling rate gradients are avoided. With this technique, only small quantities of samples can be stored on a cryosubstrate, which can be disadvantageous in applications requiring large sample volumes (e.g. in the ml range).
The object of the invention is to provide improved sample carriers for the cryopreservation of biological samples with which the disadvantages of conventional sample carriers are overcome. Sample carriers according to the invention should in particular make it possible to reproducibly set defined freezing or thawing protocols wherein temperature gradients or variations in the cooling or thawing rate inside a sample should be avoided. The sample carrier should furthermore be suitable for the preservation of large sample volumes. The object of the invention is also to provide improved methods for cryopreservation with which the disadvantages of conventional cooling or thawing protocols are avoided.